Method for controlling fermentation process

ABSTRACT

In a fermentation medium, in which an organic acid is consumed as a carbon source by a microorganism and is added to the medium at a rate to maintain a set pH value, the concentration of the acid is held within limits required for optimum fermentation conditions by varying the concentration of bicarbonate ions in the medium. The bicarbonate concentration is increased at excessive organic acid concentration by increasing the partial pressure of carbon dioxide in the fermentation vessel or by raising the set pH limit at which the organic acid is fed to the medium. The bicarbonate concentration is reduced at insufficient organic acid concentration by decreasing the partial carbon dioxide pressure in the fermentation vessel or by lowering the set pH limit.

nited States Patent [191 lliteda et al.

- METHOD FOR CONTROLLING FERMENTATION PROCESS [75] Inventors: ShigehoIkeda; Ayaaki Ishi-Zaki,

both of Kawasaki; Yoshio Hirose, Fujisawa, all of Japan [7 3] Assignee:Ajinomoto (30., Inc., Tokyo, Japan [22] Filed: Aug. 16, 1972 [21] Appl.No.: 281,043

[30] Foreign Application Priority Data 6/1971 Shiio et a1. 195/30 R Oct.16, 1973 3,121,669 2/1964 Baptist 195/118 Primary Examiner-Alvin E.Tanenholtz Attorney-Kurt Kelman et a1.

[5 7] ABSTRACT In a fermentation medium, in which an organic acid isconsumed as a carbon source by a microorganism and is added to themedium at a rate to maintain a set pH value, the concentration of theacid is held within limits required for optimum fermentation conditionsby varying the concentration of bicarbonate ions in the medium. Thebicarbonate concentration is increased at excessive organic acidconcentration by increasing the partial pressure of carbon dioxide inthe fermentation vessel or by raising the set pH limit at which theorganic acid is fed to the medium. The bicarbonate concentration isreduced at insufficient organic acid concentration by decreasing thepartial carbon dioxide pressure in the fermentation vessel or bylowering the set pl-l limit.

4 Claims, 3 Drawing Figures Patented Oct. 16, 1973 FIG. I

00 x10' (0 Im) CO x 10' (0 fm) 2 Sheets-Sheet 1 WLLAA 2'0 CULTIVATIONTIME (hours) wihv CULTIVATION TIME (hoursI ACETIC ACID CONCENTRATION(g/dI) ACETIC ACID CONCENTRATION 2 Sheets-Sheet 2 FIG. 3

ACETlC ACID CONCENTRATION (g/dl) o 8 05 CH3COOH o I I l I l l O 10 2O 3O4O 5O 6O CULTIVATION TIME (hours) METHOD FOR CONTROLLING FERMENTATIONPROCESS This invention relates to a method for controlling afermentation process, and particularly to a method for holding theconcentration of an organic acid which is continuously or intermittentlyfed to a culture medium as a carbon source within a range suitable formicrobial growth and for the desired fermentation.

Fermentation in a culture medium is known to be impaired by an excessiveconcentration of an organic acid employed as a carbon source and to beretarded by a deficiency of the carbon source. Thus it is necessary tocontrol the concentration within a fairly narrow optimum range.

It is most convenient to replenish a culture medium with an organic acidwhen the pH of the culture medium rises beyond a certain level bymicrobial consumption of organic acid. However, it is very difficult bythis method to control the concentration of organic acid within adesired range, since there are many factors influencing the pH of aculture medium other than microbial consumption of organic acid.

On the other hand, when the concentration of organic acid is accuratelycontrolled by varying the feeding rate the pH of the culture mediumcannot be controlled within a desired range. Therefore, it has been verydifficult heretofore to control both the pH of a culture medium and theconcentration of organic acid.

It has now been found that the concentration of organic acid can beaccurately controlled with a permissible change in pH of the culturemedium, when the organic acid is fed as a carbon source to afermentation vessel so as to maintain the pH of the culture medium belowa certain level and by increasing or decreasing the concentration ofbicarbonate ions.

The concentration of bicarbonate ions can be controlled by changing thepartial pressure of carbon dioxide in the fermentation vessel and/or bychanging the pH limit set for feeding organic acid.

The relationship between the concentration of organic acid and theconcentration of bicarbonate ions is illustrated in Table l byexperimental data. The experiments were carried out by the methoddescribed in Example 1.

TABLE 1 CO, (atm) HCO (m mole) CH COO (m mole) 0.017 26 162 0.021 41 1450.031 51 122 0.055 105 77 0.078 135 47 The concentration of bicarbonateions was controlled by changing the partial pressure of carbon dioxide.

The pH of the liquid culture medium was maintained within the range from7.8 to 8.0.

TABLE 2 pH of Medium HCO (m mole) CH;,COO'(m mole) 7.75 67 300 8.00 118231 8.25 210 101 8.50 374 The concentration of bicarbonate ions wascontrolled by changing the set pH limit.

The partial pressure of carbon dioxide was controlled at about 0.08 atm.

It can be seen from Tables 1 and 2 that the concentration of acetateions is decreased by increasing the concentration of bicarbonate ions,and that the concentration of bicarbonate ions is increased byincreasing the partial pressure of carbon dioxide or by changing the pHlimit to a higher level.

The fermentation process of this invention is carried out as follows:

Small amounts of organic acid solution are fed to a culture medium so asto maintain the pH below a certain level.

When the concentration of organic acid ion rises above the rangedesirable for fermentation, the concentration of bicarbonate ions isincreased by increasing the partial pressure of carbon dioxide and/orchanging the pH limit to a higher level. When the concentration oforganic acid ions is below the range desirable for fermentation, theconcentration of bicarbonate ions is decreased by decreasing the partialpressure of carbon dioxide and/or changing the pH to a lower level.

The partial pressure of carbon dioxide can be adjusted by changing theflow rate of air or the pressure of the gaseous phase in thefermentation vessel or by introducing carbon dioxide into thefermentation vessel.

The method of this invention permits the concentration of organic acidto be controlled in any fermentation process in which organic acid isemployed as a carbon source, such as the production of glutamic acid,lysine, threonine, leucine, iso leucine, phenylalanine, citric acid,a-ketoglutaric acid, protease, amylase or microbial cells.

The organic acids for the method of this invention are assimilable suchas acetic acid, fumaric acid, malic acid, gluconic acid, benzoic acid orfatty acid.

When the concentration of organic acid is controlled by the method ofthis invention, the concentration is accurately controlled with anallowable change of pH for fermentation.

EXAMPLE 1 Ten liters of culture medium was prepared to contain 3 g/dlcasein, 5 ml/dl corn steep liquor, 0.375 g/dl KH PO 0.02 g/dl MgSO 7H Oand 2 mild] soy bean protein hydrolyzate and was placed in 30 1fermentation vessel. The medium was adjusted to pH 7.0, and wasinoculated after sterilization with previously cultured Bacillus sp. AJ3205 (PERM-P 304) which is capable of producing alkaline protease.

Fermentation was carried out aerobically at 34C for 64 hours. During thefermentation, small amounts of percent acetic acid solution were fed soas to maintain a pH from 7.8 to 8.0. When the concentration of aceticacid in the medium was below 0.2 g/dl, the partial pressure of CO in thefermentation vessel was decreased by decreasing the rate of CO supplyfrom a reservoir to the fermentation vessel. When the concentration ofacetic acid was above 0.5 g/dl, the partial pressure of CO was increasedbyincreasing the rate of CO supply.

The pH of the medium, the concentration of acetic acid and the partialpressure of C0; are shown in FIG. 1. After the fermentation, 13,000units/ml alkaline protease was found in the fermentation broth.

On the other hand, when the partial pressure of CO was not controlled,acetic acid was deficient from 15 hours towlioii r s aririfiematron andwas excessive after 58 hours. Alkaline protease found in thefermentation broth was 5,800 units/ ml. (FIG. 2)

When acetic acid was fed to the medium so as to control theconcentration of acetic acid within the range from 0.2 g/dl to 0.5 g/d]according to analytical data, the pH of the medium became too low in theearlier stage and too high in the later period of the fermentation. Thealkaline protease accumulated in the fermentation broth was less than2,000 units/ml.

EXAMPLE 2 In the same fermentation as in Example 1, when theconcentration of acetic acid was below 0.15 g/dl, the pH limit forfeeding 70 percent acetic acid solution, which had originally been setat 7.8, was changed to a lower value, and when the concentration wasabove 0.5 g/dl the pH limit was changed to a higher value.

The time course of this fermentation is shown in FIG. 3. After 64 hourscultivation, 12,800 units/ml alkaline protease was found in thefermentation broth.

EXAMPLE 3 Ten liters of a culture medium was prepared to contain 2 g/dlammonium acetate, 1 g/dl sodium acetate, 1 ug/l biotin, 0.5 ml/dlsoybean protein acidhydrolyzate, 100 Lg/l thiamine 'HCl, 0.1 g/dl KH PO0.04 g/dl MgSO -7H O, mg/dl FeSO -7H O and 10 mg/dl MnSO -4H O andadjusted to pH 7.3. The medium was placed in a 30 l fermentation vessel,sterilized by steam and inoculated with previously cultured glutamicacid-producing Brevibacterium flavum 2247 (ATCC 14067). Fermentation wascarried out aerobically at 3 l .5C. During the fermentation, the pH ofmedium was controlled within the range from 7.8 to 8.0 by adding asolution containing and ammonium acetate (acetic acid mole ratioconcentration 1.0/1.1, concentration 50 percent as acetate ion). Theconcentration of acetic acid was controlled between 0.5 g/d] and 1.0g/dl by changing the partial pressure of carbon dioxide in thefermentation vessel. After 40 hours cultivation, 7.2 g/dl glutamic acidwas found in the fermentation broth. The conversion rate from aceticacid was 52 percent by weight.

When the fermentation mentioned above was carried out withoutcontrolling the partial pressure of carbon dioxide, the concentration ofacetic acid fluctuated from 0.0 to 1.5 g/dl, and 4.3 g/dl glutamic acidwas accumulated (the conversion rate was 45 percent.

When acetic acid was fed to the culture medium so as to control theconcentration of acetic acid at about 0.75 g/dl, the pH of the mediumfluctuated from 5.2 to 9.0 and the amount of glutamic acid accumulatedin the fermentation broth was 2.37 g/dl.

EXAMPLE 4 EXAMPLE 5 Ten liters ofa culture medium was prepared tocontain 2 g/dl ammonium benzoate, 0.2 g/dl KH PO.,, 0.7 g/dl K,HPO 0.01g/dl MgSO,-7H,O, 0.1 g/dl (NH,) SO,, 2 ug/l biotine, 100 .tg/lthiamine-HCl, 0.4

ml/dl corn steep liquor and 0.25 g/dl urea, placed in 30 1 fermentationvessel and sterilized by steam.

Brevibacterium lactofermentum ATCC 13869 was inoculated in the medium,and cultured aerobically at 30C. A benzoic acid solution (whichcontained 1 part of benzoic acid per 50 parts of ammonium benzoate, and17 g/dl benzoate ion) was fed to the medium so as to maintain a pH of7.5 to 7.7. The concentration of benzoic acid was controlled within therange from 0.5 g/d] to 1.0 g/dl by controlling the partial pressure ofcarbon dioxide in the fermentation vessel. After 32 hours cultivation,5.92 g/dl glutamic acid was produced in the fermentation broth. (theconversion rate from benzoic acid was 65.8 percent by weight) When theconcentration of benzoic acid was controlled by changing the pH limitoriginally set at 7.7 to a higher point or to a lower point, after 32hours cultivation, 5.51 g/dl glutamic acid was accumulated in thefermentation broth.

On the other hand, when the fermentation was carried out withoutcontrolling the concentration of benzoic acid, the concentration ofbenzoic acid fluctuated from 0.0 to 3.0 g/dl, and 2.62 g/dl of glutamicacid was accumulated.

EXAMPLE 6 Ten liters of a culture medium was prepared to contain 1.2g/dl ammonium acetate, 0.7 g/dl (NH SO.,, 0.2 g/dl KH PO 0.04 g/dl MgSO7H O, 10 mg/dl FeSO, 7H O, 10 mg/dl MnSO 4H O, 4 ml/dl soybean proteinhydrolyzate, 15 mg/dl L-threonine, 30 mgldlD L-methionine, 50ugl'l'biotine' iofigl 1 thiamine-HCl, 0.2 g/dl urea and adjusted to pH6.5. The medium was placed in a 30 1 fermentation vessel and inoculatedwith Brevibacterium flavum ATCC 21129. The fermentation was carried outaerobically at 31.5C, maintaining the pH originally within the rangefrom 7.5 to 7.7. by feeding 60 percent acetic acid solution whichcontained 1 part of acetic acid and 0.5 parts ammonium acetate.

The concentration of acetic acid was controlled within the range from0.5 to 1.0 g/dl by changing the partial pressure of carbon dioxide.After 48 hours cultivation, 6.47 g/dl lysine was accumulated in thefermentation broth.

When the concentration of acetic acid was controlled within the samerange as above by changing the pH limit, the amount of accumulatedlysine was 6.38 g/dl. The highest and the lowest set limits of pH were8.2 and 7.2 respectively.

When the concentration of acetic acid was not controlled, theconcentration fluctuated from 0.0 to 3.2 g/dl,, and the amount ofaccumulated lysine was 4.20 g/dl.

We claim:

1. In a method of microbial fermentation in which an organic acid isconsumed as a carbon source in a culture medium and replenished when thepH of the medium rises above a limit set within a predetermined range,and the concentration of said acid is controlled within an optimum rangefor successful fermentation, the improvement in controlling saidconcentration which comprises conducting the fermentation under partialcarbon dioxide pressure to provide bicarbonate ions and:

gaseous phase in contact with said medium.

3. In a method as set forth in claim ll, said concentration ofbicarbonate ions being increased and decreased by varying the pH.

4. In a method as set forth in claim ll, said organic acid being aceticacid, fumaric acid, malic acid, gluconic acid, benzoic acid, or a fattyacid.

2. In a method as set forth in claim 1, said concentration ofbicarbonate ions being increased and decreased by varying the partialpressure of carbon dioxide in a gaseous phase in contact with saidmedium.
 3. In a method as set forth in claim 1, said concentration ofbicarbonate ions being increased and decreased by varying the pH.
 4. Ina method as set forth in claim 1, said organic acid being acetic acid,fumaric acid, malic acid, gluconic acid, benzoic acid, or a fatty acid.